The proposed research program will be a continuation of the development and utilization of magnetic resonance techniques to investigate the interactions of substrates with enzymes which catalyze reactions of nucleotides. The objective is to determine structures and conformations of enzyme-substrate complexes in solution in order to elucidate the molecular basis of substrate specificity and the mechanism of enzymatic catalysts. We shall extend the 31P-NMR studies of enzyme-bound substrates initiated with arginine kinase and creatine kinase to adenylate kinase. The Beta-P resonance of enzyme-bound ADP is about 2-3 ppm from that of the unbound form due to an apparent pK shift. The apparent pK of 7.5 is similar to the pK observed in the rate of the forward reaction of arginine kinase. By extending such studies to adenylate kinase where the proton NMR of the protein can be observed, it should be possible to correlate the change in the substrate 31P-NMR with that of the changes in the amino acid residue to which it is responding as a function of pH. Furthermore it is planned to use the paramagnetic metal ion activator Mn(II) with the substrates ATP and ADP and the inhibitor Ap5A to measure T1 of C-2 proton of His 36 at the active site to determine distances. The structure of enzyme-bound MgATP with nucleotidyl transferring enzymes, aminoacyl tRNA synthetases, will be compared with kinases, phosphoryl transferring enzymes, to delineate differences. Since the chemical shift of the Beta-P of ATP is very sensitive to coordination with Mg, these experiments should give the desired result. Finally it is hoped that it will be possible to interpret results obtained on proton NMR of tRNA-Met/f and tRNA-Met/M bound to cognate aminoacyl tRNA synthetase in terms of specificity of binding. BIBLIOGRAPHIC REFERENCES: Magnetic Resonance Study of the Three-Dimensional Structure of Creatine Kinase-Substrate Complexes. Implications for Substrate Specificity and Catalytic Mechanism, Alan C. McLaughlin, John S. Leigh, Jr. and Mildred Cohn, J. Biol. Chem. (1976) 251, 2777-2787. Metal Ion Effects on Structure of t-RNA and Its Interaction with Aminoacyl t-RNA Synthetase, Mildred Cohn, Abstract, American Chemical Society Meeting, New York, April, 1976.